Evaluation of NKp46 expression and cytokine production of decidual NK cells in women with recurrent pregnancy loss

Abstract Purpose NKp46, a receptor on NK cells, is involved in cytotoxicity and cytokine production. The authors aimed to evaluate the effect of NKp46 on decidual NK (dNK) cells during pregnancy and whether it can be a marker for immunological abnormalities in women with recurrent pregnancy loss (RPL). Methods Flow‐cytometric analysis was made to assess NKp46 expression and intracellular cytokine production of dNK cells. The proportion of NKp46+ dNK cells was analyzed among RPL patients who aborted karyotypically normal pregnancies and those who either aborted karyotypically abnormal pregnancies or without genetic studies, and controls who were going through the induced abortion. Results The %NKp46+ and %NKp46bright dNK cells were significantly lower in the RPL women who aborted karyotypically normal pregnancies than in the control group. The %NKp46bright dNK cells were significantly correlated with the NK1/NK2 ratio of dNK cells. The %NKp46+ dNK cell cutoff for RPL with immunological abnormalities was determined by the ROC curve analysis. In women with the low %NKp46+ dNK, NK1/NK2 ratios were significantly higher than those with the high. Conclusion RPL patients with an immunological abnormality have decreased NKp46 expression and NK1 shift in dNK cells. NKp46 expression could be a marker for RPL of immunological abnormalities.

remodeling, angiogenesis, and preventing an excessive trophoblast invasion. Contrarily, dysregulated cytotoxic immune effectors may disturb pregnancy and induce pregnancy failures. 3,5,6 Apart from cytotoxic immune effectors, regulatory T (Treg) cells also increase in early pregnancy. Treg cells are known to inhibit proliferation and cytokine production of both CD4 + and CD8 + T cells, immunoglobulin production by B cells, the cytotoxic activity of natural killer (NK) cells, and maturation of dendritic cells (DCs), resulting in the induction of tolerance. 7,8 The most abundant cells in the uterine endometrium are NK cells. The number of NK cells dramatically increases in the secretory phase and during early pregnancy 9 to establish and maintain pregnancy. According to fluorescence staining intensity, NK cells express the CD56 receptor and can be subdivided into CD56 dim and CD56 bright cells. Approximately 90% of uterine NK (uNK) cells are CD56 bright cells, mainly involved in cytokine production, 10 whereas approximately 90% of peripheral blood NK (pNK) cells are CD56 dim cells, involved in cytotoxicity. 11 It has been reported that CD56 dim uNK cells increase in patients with RPL. 12 NK cells express different kinds of receptors on their surface.
NKp46, a natural cytotoxicity receptor (NCR), is involved in NK cell activation and functions in both cytotoxicity and cytokine production. 13 NKp46 is a 46-kDa type 1 membrane glycoprotein belonging to the immunoglobulin superfamily. It has two C2-type Ig-like domains in the extracellular portion and is associated with CD3ζ and FcεRIγ. 14 When it recognizes and binds to its ligand, immunoreceptor tyrosine-based activation motifs (ITAMs) are phosphorylated. They recruit Zap-70 or SYK, leading to a cascade of reactions that ends with the intracellular release of calcium, inducing cytotoxicity and cytokine release. 13 Hemagglutinin, a surface protein on influenza A and parainfluenza viruses, is an extrinsic ligand of NKp46. 15 Intrinsic ligands of NKp46 are present in murine myeloma cell lines, human nevi, and melanoma cells.
Not only that, vimentin may be an intrinsic ligand. 16 Vimentin, a 57-kDa intermediate filament protein, is used as a cell differentiation marker. It is also expressed in the uterine endometrium and utilized to diagnose and grade cervical or endometrial cancer. NK cells kill activated CD4 + T cells through the NKp46/vimentin pathway. 17 However, the role of the NKp46/vimentin pathway in reproduction has not been elucidated yet. In the case of uterine infection, NKG2A-mediated negative signals, which control NKp46-mediated cytolytic function, might be abrogated by viral immune evasion mechanisms, leading to the absence or diminished expression of its HLA-E-specific ligand, 18 and the upregulation of the NKp46 specific ligand. Such NKp46-mediated cytotoxic activity and the NKp30mediated secretion of inflammatory cytokines by decidual NK (dNK) may decrease the number of infected uterine cells. 19 Recently, we reported that the numbers of NKp46 + CD16 − NK cells were low in patients with higher CD56 dim /CD16 + NK cells, accompanied by an NK2 shift. 20 We have also reported that NKp46 dim NK cells may be involved in NK cell cytotoxicities, whereas NKp46 bright NK cells may be involved in cytokine production, suggesting that NKp46 could be a predictive marker for immune tolerance in pregnancy. 21 There are two types of NK cells: NK1 cells producing inflammatory (Type 1) cytokines, such as IFNγ and TNFα, and NK2 cells producing anti-inflammatory (Type 2) cytokines, such as IL-4 and IL-10. 22 NK cells show polarities in their cytokine secretion profiles, comparable to the polarities of T helper (Th) cells. 23 There is an increase in type 2 cytokine production in the uterus in a healthy pregnancy, called the NK2 shift 24 or Th2 shift. 25 Decreased type1 inflammatory response protects the fetus, while NK1 shift has been reported in women with RPL and recurrent implantation failure (RIF) after in vitro fertilization and embryo transfer cycles. 26 We have previously reported that NKp46 expression was low on the surface of pNK and/or uNK cells in women with various forms of reproductive failures, such as RPL and RIF. [27][28][29] Besides, we have also reported that low expression of activating receptors on NKp46 + uNK cells is more prevalent in high-risk women. 20 Thus, NKp46 plays an important role in reproduction through cytotoxicity and cytokine production. However, the detailed mechanism remains unknown and NKp46 expression on dNK cells in patients with RPL has not been investigated. Therefore, we aim to explore the role of NKp46 + dNK cells in patients with RPL and whether NKp46 can be used to detect RPL with immunological abnormalities by analyzing the relationship between the expression of NKp46 and cytokine production of dNK cells.

| Ethical approval and study participants
We enrolled patients at the Department of Obstetrics and Gynecology, Hyogo Medical University Hospital, between April 2018 and December 2020. All participants provided written, informed consent before enrolling in the study, approved by the Hyogo Medical University Institutional Review Board, and complied with the Declaration of Helsinki (2013). dNK cells were collected from 43 women undergoing dilatation and curettage (Table 1), including women with two or more pregnancy losses (the RPL group, n = 31) and those who had an induced abortion due to medical conditions or abnormal fetal karyotype (the control group, n = 12). Patients who had an artificial abortion for maternal protection or who had an abortion for the first time owing to abnormal fetal karyotype were put in the control group. The RPL group was subdivided into two subgroups; those who had abortions with karyotypically normal pregnancy (n = 11) and those with karyotypically abnormal pregnancy or without genetic studies (n = 20; including 11 patients who did not perform chorionic karyotyping).

| Measurement of surface antigens on dNK cells
Surface antigens on dNK cells were stained using the following monoclonal antibodies (Table S1) (Table   S1B).
One-way analysis of variance (ANOVA) was used to compare the distribution of age, number of pregnancies, deliveries, miscarriages, and pregnancy days among the three groups; then, Tukey's test was performed between the two groups. Data are presented as the mean ± standard deviation (SD). Differences in surface antigenic expression, intracellular cytokine production, and the NK1/ NK2 cytokine ratios among the three groups were analyzed using   Table 1 shows the obstetrical histories of the study patients and controls. The number of pregnancies in the RPL group without karyotypically normal pregnancies was significantly higher than in the control group (p < 0.05). The number of previous deliveries was significantly lower in the RPL with karyotypically normal pregnancies than in the RPL without karyotypically normal pregnancies (p < 0.05). The number of spontaneous abortions in the RPL group with and without karyotypically normal pregnancies was significantly higher than in the control group (p < 0.05, respectively). The number of induced abortions in the RPL group with karyotypically normal pregnancies was significantly lower than in the control group (p < 0.05). There was no significant difference in gestational days at abortion among the three groups.

| CD16 and CD56 co-expression on dNK cells
Representative dot-plots of CD16 and CD56 co-expressing dNK cells are shown in Figure 1E,F. The %CD16 + /CD56 dim and %CD16 − / CD56 bright cells were not significantly different among the three groups ( Table 2).

| NKp46 expression on dNK cells
Representative dot-plots of NKp46 expressed on dNK cells of each group are shown in Figure S1A-C. The %NKp46 + dNK cells in the  Figure 2B), with no significant difference between the RPL group without karyotypically normal pregnancies and the control group. The differences in the %NKp46 dim dNK cells among the three groups ( Figure 2C) were insignificant.

| Cytokine production and NK1/NK2 ratios of dNK cells
Representative dot-plots of cytokine production of dNK cells are shown in Figure 1H-L. There were no significant differences in the percentages of TNFα, IFNγ, IL-4, IL-10, or TGF-β1 producing dNK cells (Table 3A) or in the TNFα/IL-4, IFNγ/IL-4, TNFα/IL-10, and IFNγ/IL-10 producing CD56 bright dNK cell ratios among the three groups (Table 3B). CD56 bright dNK cells, but not with TNFα/IL-4 or IFNγ/IL-4 ratios of CD56 bright dNK cells ( Figure S2, Table S2B). There was no significant correlation between the %NKp46 + dNK cells and the NK1/NK2 ratios of CD56 bright dNK cells (Table S2B).  p < 0.05; Figure 3Ad). The percentages of TNFα, IFNγ, IL-10, or TGF-β1 producing CD56 bright dNK cells were comparable between the low NKp46 bright and high NKp46 bright groups (Figure 3Ab-   respectively (p < 0.05; Figure 4Ad-f). The percentages of TNFα-, or IFNγ-producing CD56 bright dNK cells were comparable between the low and high NKp46 + groups (Figure 4Aa-b) Figure 4Bb,d). The TNFα/IL-4 and TNFα/IL-10 ratios were comparable between the low and high NKp46 + groups (Figure 4Ba,c).  Red bars show the low NKp46 + group. Gray bars show the high NKp46 + group. Differences between the two groups were analyzed using the Mann-Whitney U-test; differences were considered significant at p < 0.05

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NKp46 bright and NKp46 + cells in the RPL group with karyotypically normal pregnancies. The NK1/NK2 ratios correlate with the percentages of NKp46 bright cells rather than NKp46 + cells, suggesting that %NKp46 bright has a function in cytokine production (Table S2 and Figure S2). NKp46 bright cells are related to cytokine production, and in the RPL group with karyotypically normal pregnancies, the reduction of NKp46 bright cells is thought to cause abnormal cytokine production (NK1 shift), leading to miscarriage. However, as shown in During the cytokine production analysis of CD56 bright dNK cells (Table 3), higher TNFα and IFNγ production and lower IL-4, IL-10, and TGF-1β production were observed in the RPL group with karyotypically normal pregnancies compared with the control group, although differences were not significant. NK1 cytokines such as TNFα and IFNγ contribute to a successful pregnancy with TGF-β1 by participating in spiral artery remodeling and establishing vascular connections between the fetus and the mother; however, excessive inflammatory cytokine production can be harmful to the placenta. Therefore, a balance between NK1 and NK2 cytokine production and a timely shift to NK2 cytokine production is critical to a successful pregnancy. 23 In this study, TNFα/IL-4, TNFα/IL-10, IFNγ/IL-4, and IFNγ/IL-10 producing dNK cell ratios appeared to shift to NK1 in the RPL group with karyotypically normal pregnancies.

ACK N OWLED G M ENTS
The authors would like to thank all patients who participated in this research. This work was supported by JSPS KAKENHI (Grant Number JP16K11078 and JP21K09504).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

H U M A N R I G HT S S TATE M E NT S A N D I N FO R M E D CO N S E NT
This study was approved by the institutional review board of the Hyogo Medical University (IRB number 2871). Human rights statements and informed consent: all procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and its later amendments. Informed consent was obtained from all patients being included in the study.

A N I M A L R I G HTS
This article does not contain any studies with animal subjects performed by any authors.